Identification of reduced fluoroquinolone susceptibility in Escherichia coli: a herald for emerging resistance.

Abstract

Sir, Target changes via alterations of DNA gyrase and/or topoisomerase IV, and reduced intracellular drug accumulation, either by reduced drug permeability and/or increased efflux activity, are the two major mechanisms that mediate fluoroquinolone resistance. Resistance to quinolones in Escherichia coli appears to be caused mainly by alterations in the gyrA gene of the DNA gyrase and in the parC gene of the topoisomerase. The mutations are located at the N-terminus of the GyrA protein (residues 67–106 in E. coli) in the quinolone resistance-determining region (QRDR). A similar QRDR has also been reported in parC. We have reported as part of a national surveillance programme in Taiwan 526 E. coli isolates that were classified as fluoroquinolone susceptible according to the NCCLS guidelines. However, the distribution of zone diameters of ciprofloxacin indicated that these isolates consisted of two sub-populations with susceptibility and reduced susceptibility. From five of 44 hospitals surveyed, 193 E. coli isolates were selected for further study. Of these 193 isolates, 20 (10.4%) were with resistance, 44 (22.8%) with reduced susceptibility and 129 (66.8%) with susceptibility with zone diameters of ciprofloxacin 15, 16–29 and 30 mm, respectively, The 20 isolates with resistance, 44 isolates with reduced susceptibility and a random sample consisting of 17 E. coli isolates with susceptibility were assessed for point mutations in the QRDRs of gyrA and parC to determine the genetic bases of fluoroquinolone susceptibility. Based on their mutations in gyrA and parC, the 81 isolates were divided into 13 classes (Table). All resistant isolates in our study had two mutations in gyrA and at least one additional mutation in parC (Table, classes 1–5). A major type of combined mutations, Ser-83 Leu, Asp-87 Asn in gyrA and Ser-80 Ile in parC, was found in 17 resistant isolates (85%). Although single mutations in gyrA have previously been associated with reduced susceptibility to fluoroquinolones in E. coli, the molecular epidemiology of mutations in both gyrA and parC and their associations with reduced susceptibility have not been delineated. All isolates with reduced susceptibility in our study had one mutation in gyrA (Table, classes 6–12). Ser-83 Leu was a major mutation found in the isolates with reduced susceptibility, detected in 39 strains (87%). Isolates with double mutations in gyrA and parC (classes 6–8) had higher MIC90s of fluoroquinolones (0.75 and 2 mg/L of ciprofloxacin and ofloxacin, respectively) than isolates with only one mutation in gyrA (class 9, 0.25 and 1 mg/L of ciprofloxacin and ofloxacin, respectively). These data indicate that additional mutations in parC result in slight increases in MICs, consistent with the results reported for Neisseria gonorrhoeae. No strain possessed a parC mutation without the simultaneous presence of a gyrA mutation. These findings are consistent with fluoroquinolone resistance arising in a multi-step fashion with mutations in gyrA occurring as the first step. Isolates with zone diameters of ciprofloxacin of 15, 16–20 and 21 mm are considered as resistant, intermediate and susceptible, respectively, to ciprofloxacin according to current NCCLS resistance breakpoints. A major concern related to finding a large number of E. coli (22.8%) with reduced susceptibility, especially those with zone diameters of ciprofloxacin between 21 and 29 mm, is the fact that these isolates are unlikely to be detected using current NCCLS guidelines. Increased clinical failures have been reported when human infections caused by Salmonella spp. with reduced susceptibility are treated with fluoroquinolones. Owing to similar concerns over treatment efficacy, the breakpoint for the definition of resistance to fluoroquinolones in N. gonorrhoeae was recently adjusted as follow: isolates with zone diameters of ciprofloxacin 27, 28–40 and 41 mm are interpreted as resistant, intermediate and susceptible, respectively, to ciprofloxacin. Antimicrobial susceptibility test results should not only guide clinicians in determining the most appropriate therapy for their patients, but also assist efforts to monitor and control the spread of resistance. We therefore recommend that current NCCLS breakpoints for fluoroquinolone resistance in Enterobacteriaceae, such as E. coli and Salmonella spp., be modified according to our findings: Correspondence Journal of Antimicrobial Chemotherapy (2001) 48, 931–942